The purpose of this program is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. Earlier studies in this laboratory examined the ability of C. diphtheriae to transport and utilize heme and heme proteins as iron sources. These studies identified and characterized the hmuO gene, which encodes a heme oxygenase involved in the removal of iron from heme, and an ABC heme transporter required for heme transport and heme iron utilization. Current studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) the development of genetic tools in C. diphtheriae, 3) the characterization of a DtxR homolog in C diphtheriae termed MntR, and 4) the construction and analysis of a DtxR repressor titration library that was used to identify ten novel DtxR binding sites on the C. diphtheriae chromosome. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. Chromosomal deletions in both chrA and chrS were constructed in C. diphtheriae and were shown to significantly reduce, although not totally abolish, the heme-dependent activation at the hmuO promoter. This finding suggests that additional regulatory factors are involved in the regulation of hmuO. A homolog of dtxR, mntR, was identified from the recently completed C. diphtheriae genome. The mntR gene was cloned and was shown to be the terminal gene in a five gene operon that also encoded a putative ABC-metal transporter. The product of mntR was shown to regulate expression of this operon by a Mn-dependent mechanism. The diphtheria toxin repressor, DtxR, is a global iron-dependent regulatory protein in Corynebacterium diphtheriae that controls gene expression by binding to 19-bp operator sequences. To further define the DtxR regulon in C. diphtheriae, a DtxR repressor titration assay (DRTA) was developed and used to identify 10 previously unknown DtxR binding sites. Open reading frames downstream from seven of the newly identified DtxR binding sites are predicted to encode proteins associated with iron or heme transport. A putative siderophore biosynthesis and transport operon located downstream from one of the DtxR binding sites, designated sid, is similar to the yersiniabactin synthesis and uptake genes encoded on the Yersinia pestis high-pathogenicity island. The siderophore biosynthetic genes in the sid operon contained a large deletion in the C. diphtheriae C7 strain, but the sid genes were unaffected in four clinical isolates that are representative of the dominant strains from the recent diphtheria epidemic in the former Soviet Union. These results indicate that C. diphtheriae contains at least 18 DtxR binding sites, and that DtxR may affect the expression of as many as 40 genes. Characterization of the iron regulon in Bacillus anthracis. Anthrax is a severe disease caused by the gram-positive bacterium Bacillus anthracis. The organism infects humans and many other animals and the only identified virulence factors are anthrax toxin and a glutamic acid capsule, both of which are encoded on large plasmids. Inhalation anthrax, the most severe form of the disease, is a systemic infection in which the organism spreads to the lymph nodes and then into the blood where it is able to replicate to levels as high as 100 million bacteria / ml. Factors encoded on the chromosome have also been implicated in virulence, since certain strains lacking the virulence plasmids are still capable of causing disease in immunized animals. The future development of these "vaccine resistant" strains as bioterrorism weapons is of major concern, and stresses the importance of establishing a fundamental understanding of the molecular mechanisms involved in the pathogenesis of this bacterium. For many bacterial pathogens, the ability to survive in the human host requires the acquisition of the essential element iron. Mechanisms involved in the acquisition of iron have been shown to be important for the virulence of numerous bacterial pathogens, including organisms that replicate in the blood, where much of the available iron is sequestered by host iron compounds such as transferrin and hemoglobin (in erythrocytes). No investigations have examined the mechanism by which B. anthracis acquires iron during growth in vivo or in vitro. This is an important area of study that has not been explored, and in this proposed research, I intend to test the hypothesis that virulence factors involved in the transport and utilization of host iron compounds are present in B. anthracis. Our long-term interest is to identify surface proteins that are involved in the utilization of host iron compounds, and determine if these proteins may be useful vaccine candidates. We have developed a low-iron minimal medium to cultivate B. anthracis and have used this medium to show that the bacterium could use a variety of host compounds as essential iron sources, including heme, hemoglobin, ferritin, transferrin, and lactoferrin. In the past year we have independently developed a method for constructing site directed mutations in the chromosome of B. anthracis. This technique has been used to construct mutations in genes involved in the biosynthesis of the B. anthracis siderophore. The siderophore mutations resulted in strains that had diminished production of iron chelating activity, but nevertheless still made detectable quantities of an extracellular iron-chelating compound. These findings suggested that B. anthracis may produce multiple siderophores. In an attempt to identify proteins involved in the utilization of transferrin and lactoferrin as iron sources, we have used affinity chromatography to enrich for iron-regulated membrane proteins that bind to transferrin and lactoferrin. Several proteins that have the ability to bind to transferrin and lactoferrin were partially purified using this technique and the amino acid sequence of their N-terminal is currently being determined. The N-terminal sequence obtained from these proteins will be used to identify the genes that encode these various proteins. Genes and their associated products that appear to be directly involved in the utilization of either transferrin or lactoferrin as iron sources will be examined in greater detail. Identification and characterization of virulence determinants in C. diphtheriae. The purpose of this project is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. Earlier studies in this laboratory examined the ability of C. diphtheriae to transport and utilize heme and heme proteins as iron sources. These studies identified and characterized the hmuO gene, which encodes a heme oxygenase involved in the removal of iron from heme, and an ABC heme transporter required for heme transport and heme iron utilization. Current studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) the development of genetic tools in C. diphtheriae, 3) the characterization of a DtxR homolog in C diphtheriae termed MntR, and 4) the construction and analysis of a DtxR repressor titration library that was used to identify ten novel DtxR binding sites on the C. diphtheriae chromosome. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. Chromosomal deletions in both chrA and chrS were constructed in C. diphtheriae and were shown to significantly reduce, although not totally abolish, the heme-dependent activation at the hmuO promoter. This finding suggests that additional regulatory factors are involved in the regulation of hmuO. A homolog of dtxR, mntR, was identified from the recently completed C. diphtheriae genome. The mntR gene was cloned and was shown to be the terminal gene in a five gene operon that also encoded a putative ABC-metal transporter. The product of mntR was shown to regulate expression of this operon by a Mn-dependent mechanism. The diphtheria toxin repressor, DtxR, is a global iron-dependent regulatory protein in Corynebacterium diphtheriae that controls gene expression by binding to 19-bp operator sequences. To further define the DtxR regulon in C. diphtheriae, a DtxR repressor titration assay (DRTA) was developed and used to identify 10 previously unknown DtxR binding sites. Open reading frames downstream from seven of the newly identified DtxR binding sites are predicted to encode proteins associated with iron or heme transport. A putative siderophore biosynthesis and transport operon located downstream from one of the DtxR binding sites, designated sid, is similar to the yersiniabactin synthesis and uptake genes encoded on the Yersinia pestis high-pathogenicity island. The siderophore biosynthetic genes in the sid operon contained a large deletion in the C. diphtheriae C7 strain, but the sid genes were unaffected in four clinical isolates that are representative of the dominant strains from the recent diphtheria epidemic in the former Soviet Union. These results indicate that C. diphtheriae contains at least 18 DtxR binding sites, and that DtxR may affect the expression of as many as 40 genes. This project incorporates FY2002 projects 1Z01BJ004003-08 and 1Z01BJ004007-02.